The global pandemic of dengue fever has escalated dramatically in recent decades, accompanied by an increase in the more severe manifestation of the disease, dengue hemorrhagic fever (DHF). Mosquito-borne dengue virus (genus Flavivirus), the agent of dengue disease, currently causes an estimated 100 million infections per year in more than 100 countries resulting in 500,000 cases of DHF and 25,000 deaths. Since neither a licensed vaccine nor antiviral therapy is available to control dengue disease, new methods to control this emerging disease are urgently needed. However the rational development of these methods requires more information about the evolution of dengue virus-vector interactions than is currently available. Specifically, it is crucial to identify the targets of vector-driven selection in the dengue virus genome, i.e. those regions of the genome that affect the specificity and efficiency with which dengue virus infects its mosquito vectors. The 80 species of flaviviruses group into three genetic lineages that differ in their mode of transmission: (a) mosquito-borne, (b) tick-borne and (c) no known vector (assumed to be directly transmitted). Comparisons among these lineages have identified regions of sequence that show conserved differences between vector-borne and non-vector-borne viruses and between tick-borne and'mosquito-borne viruses. Three hypotheses to explain this pattern of conserved differences are: (i) adaptations for mode of transmission, (ii) adaptations for different sets of vertebrate hosts, (iii) non-adaptive phylogenetic inertia. The proposed research will distinguish among these hypotheses by experimentally manipulating regions of conserved difference in the dengue virus 3' untranslated region to create viruses in which the region had been (i) deleted, (ii) replaced with homologous sequence from tick-borne Langat virus, (iii) replaced with homologous sequence from directly-transmitted Modoc virus. Each mutant virus will be tested for its ability to infect mosquito and mammalian model systems.